Induction of β cell differentiation in human cells by stimulation of the GLP-1 receptor

ABSTRACT

The present invention provides methods for inducing insulin gene expression in cultured pancreas cells, the method comprising contacting a culture of endocrine pancreas cells expressing a PDX-1 gene with a GLP-1 receptor agonist, wherein the cells have been cultured under conditions such that the cells are in contact with other cells in the culture, thereby inducing insulin gene expression in the ceils. The invention also provides methods of treating a diabetic human subject using the methods of the invention.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/160,336, filed May 30, 2002, now U.S. Pat. No. 6,884,585,issued Apr. 26, 2005, which is a continuation of U.S. patent applicationSer. No. 09/522,376, filed Mar. 10, 2000, now U.S. Pat. No. 6,448,045,issued Sep. 10, 2002.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. 5 R01DKK55283-02 and 5 R01 DK55065-02, awarded by the National Institutes ofHealth. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Transplantation of cells exhibiting glucose-responsive insulin secretionhas the potential to cure diabetes. However, this approach is limited byan inadequate supply of cells with that property, with is exhibited onlyby pancreatic β-cells. The development of expanded populations of humanβ-cells that can be used for cell transplantation is therefore a majorgoal of diabetes research (D. R. W. Group, “Conquering diabetes: astrategic plan for the 21st century” NIH Publication No. 99-4398(National Institutes of Health, 1999)). A number of alternativeapproaches are being pursued to achieve that goal, including usingporcine tissue as a xenograft (Groth et al., J Mol Med 77:153-4 (1999)),expansion of primary human β-cells with growth factors and extracellularmatrix (Beattie et al., Diabetes 48:1013-9 (1999)), and generation ofimmortalized cell lines that exhibit glucose-responsive insulinsecretion (Levine, Diabetes/Metabolism Reviews 1: 209-46 (1997)).

Although there has been great interest in using porcine islets, they aredifficult to manipulate in vitro and concerns have been raised aboutendogenous and exogenous xenobiotic viruses being transmitted to graftrecipients (Weiss, Nature 391:327-8 (1998)). With primary human β-cells,entry into the cell cycle can be achieved using hepatocyte growthfactor/scatter factor (“HGF/SF”) plus extracellular matrix (“ECM”)(Beattie et al., Diabetes 48:1013-9 (1999), Hayek et al., Diabetes44:1458-1460 (1995)). However, this combination, while resulting in a2-3×10⁴-fold expansion in the number of cells, is limited by cellularsenescence and loss of differentiated function, particularly pancreatichormone expression (Beattie et al., Diabetes 48:1013-9 (1999)).

Immortalized cell lines from the human endocrine pancreas have beencreated to develop β-cell lines that exhibit glucose responsive insulinsecretion (Wang et al., Cell Transplantation 6:59-67 (1997), Wang etal., Transplantation Proceedings 29:2219 (1997), Halvorsen et al.,Molecular and Cellular Biology 19:1864-1870 (1999)). The cell lines aremade by infecting primary cultures of cells from various sourcesincluding adult islets, fetal islets, and purified β-cells, with viralvectors expressing the potent dominant oncogenes such as SV40 T antigenand H-ras^(val12) (Wang et al., Cell Transplantation 6:59-67 (1997),Wang et al., Transplantation Proceedings 29:2219 (1997), Halvorsen etal., Molecular and Cellular Biology 19:1864-1870 (1999); see also U.S.Pat. No. 5,723,333). The combined effect of those oncogenes is totrigger growth factor-independent and extracellular matrix(ECM)-independent entry into the cell cycle, as well as to prolong thelifespan of the cells from 10-15 population doublings or primary cellsto approximately 150 doubling for the oncogene-expressing cells(Halvorsen et al., Molecular and Cellular Biology 19:1864-1870 (1999)).Further introduction of the gene encoding the hTRT component oftelomerase results in immortalization, allowing the cells to be grownindefinitely (Halvorsen et al., Molecular and Cellular Biology19:1864-1870 (1999)).

Although the cell lines grow indefinitely, they lose differentiatedfunction, similar to growth-stimulated primary β-cells. Methods ofstimulating differentiation of the cell lines into insulin-secretingβ-cells are therefore desired. Such cells could then be transplanted invivo as a treatment for diabetes.

SUMMARY OF THE INVENTION

Induction of β-cell differentiation in cultured human β-cells wasachieved by stimulating multiple signaling pathways, including thosedownstream of the homeodomain transcription factor PDX-1, cell-cellcontact, and the glucagon-like peptide-1 (GLP-1) receptor. Synergisticactivation of those pathways resulted in differentiation of the culturedhuman β-cells, which initially express no detectable pancreatichormones, into fully functional β-cells that exhibit glucose-responsiveinsulin secretion. Furthermore, these cells can be transplanted in vivoand demonstrate glucose-responsive expression of insulin. The ability togrow unlimited quantities of functional human β-cells in vitro providesthe means for a definitive cell transplantation therapy for treatment ofdiabetes.

In one aspect, the present invention provides a method for inducinginsulin gene expression in cultured endocrine pancreas cells, the methodcomprising the steps of (i) expressing a PDX-1 gene in cells that havebeen cultured under conditions such that the cells are in contact withother cells in the culture; and (ii) contacting the cells with a GLP-1receptor agonist, thereby inducing insulin gene expression in the cells.

In another aspect, the present invention provides a method ofidentifying a compound that modulates β-cell function, the methodcomprising the steps of contacting cells made by the method describedabove with the compound and determining the effect of the compound onβ-cell function.

In another aspect, the present invention provides a stable culture ofendocrine pancreas cells, wherein the cells are in contact with othercells in the culture, wherein the cells express a PDX-1 gene, andwherein insulin gene expression is stimulated in the cells when exposedto an effective amount of a GLP-1 receptor agonist.

In another aspect, the present invention provides a method of treating adiabetic subject by providing to the subject cells that secrete insulinin response to glucose, the method comprising the steps of: (i)contacting a culture of endocrine pancreas cells expressing a PDX genewith a GLP-1 receptor agonist, wherein the cells have been culturedunder conditions such that the cells are in contact with other cells inthe culture; and (ii) administering the cells to the subject, therebyproviding to the subject cells that secrete insulin in response toglucose.

In one embodiment, the GLP-1 receptor agonist is a GLP-1 analog or hasan amino acid sequence of a naturally occurring peptide. In anotherembodiment, the GLP-1 receptor agonist is GLP-1, exendin-3, orexendin-4.

In one embodiment, the cells are cultured as aggregates in suspension.

In one embodiment, the PDX-1 gene is endogenous to the cells. In anotherembodiment, the PDX-1 gene is recombinant.

In one embodiment, the cells are human cells. In another embodiment, thecells are βox5 cells. A deposit of the βox5 cells, which are humanpancreatic cells, was made on Jul. 19, 2001 under accession numberPTA-3532 at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209.

In one embodiment, the cells express a recombinant oncogene. In anotherembodiment, the cells express a recombinant oncogene. In anotherembodiment, the cells express a recombinant oncogene. In anotherembodiment, the cells express a recombinant telomerase gene.

In one embodiment, the diabetic subject is a human. In anotherembodiment, the subject has Type I insulin dependent diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Flow cytometric analysis of FAD autofluorescence in a singlecell suspension of human islets used to develop the βlox5 cell line.

FIG. 2: RT-PCR analysis of pancreatic hormone gene expression in βlox5.The conditions tested were βlox5 cells grown in monolayer culture, βlox5cells infected with a retroviral vector expressing PDX-1, βlox5 cellsgrown as three-dimensional aggregates, and βlox5 cells treated withexendin-4. FIG. 2(A) insulin; FIG. 2(B) quantitative RT-PCR analysis ofinsulin gene expression; FIG. 2(C) other pancreatic hormones.Aggregation of cells into three-dimensional clusters and the retroviralvector expressing PDX-1 have been described previously (Itkin-Ansari etal., submitted). Exendin-4 (Sigma) was used at a concentration of 10 nM.RT-PCR for insulin, somatostatin, glucagon, and IAPP have been describedpreviously (Itkin-Ansari et al., submitted). Quantitative RT-PCR wasdone by interpolation from a standard curve constructed using a plasmidcontaining the human insulin cDNA.

FIG. 3: Analysis of transcription factors expressed in βlox5 cells. FIG.3(A) electrophoretic mobility shift assay (EMSA) of PDX-1. EM5A forPDX-1 was performed using a probe derived from the human insulinpromoter A5 element. FIGS. 3(B&C) RT-PCR analysis of BETA2 and Pax6.FIG. 3(D) Western blot analysis of CREB. FIG. 3(E) EMSA of RIPE3b.

FIG. 4. Insulin protein in induced βox5 cells. FIG. 4(A-C) Insulinimmunohistochemistry; FIG. 4(D) Insulin western blot analysis ofconditioned medium.

FIG. 5. Analysis of glucose-responsive insulin secretion. FIG. 5(A)RT-PCR for glucokinase. FIG. 5(B) Radioimmunoassay for insulin secretedfrom induced βlox5 cells grown in culture medium containing increasingconcentrations of glucose. Induced βlox5 cells were cultured in DMEcontaining a single concentration of added glucose for one hour. Mediumwas harvested and assayed for insulin by RIA.

FIG. 6. RIA of c-peptide in nude mice transplanted with induced βlox5cells.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

Beginning with a cell line that exhibited few β-cell characteristics,despite having been originally derived from human β-cells, completeβ-cell function has been successfully induced in vitro. In addition,thee cells have been transplanted into mice and are shown to produceinsulin in a glucose responsive manner. Induction of β-celldifferentiation in cells such as βlox5 cells requires three inducingfactors, PDX-1, cell-cell contact, and GLP-1 receptor agonists such asexendin-4. Cultured cell lines such as TRM-6 (δ-cell lineage) and βlox5(β-cell lineage) are powerful tools that should allow identification ofthe full complement of genes that are required for endocrine celldevelopment and function. Furthermore, the requirement for multipleinteracting inducing factors provides an opportunity to study howdifferent signal transduction pathways interact with one another tocontrol a complex differentiation program.

The availability of an unlimited source of functional human β-cells hasimportant implications for diabetes. One straightforward application isin exploring aspects of β-cell biology that would benefit from anunlimited, homogeneous source of cells. High-throughput screening fornew diabetes drugs is one such application. The cells of the inventioncan be used, e.g., to screen for small molecule or macromolecule GLP-1receptor agonists or other compounds that enhance insulin expression. Inaddition, cells such as those described herein can be used in a celltransplantation therapy for diabetes. Cells that express PDX-1 and arein cell-to-cell contact with other cells are stimulated with a GLP-1receptor agonist, as described herein. Such cells are then transplantedinto a suitable mammalian host, preferably a human. Such cells exhibitglucose-responsive insulin secretion in vivo.

In one embodiment, cultured cells useful in the practice of theinvention express one or more oncogenes, such as SV40 T antigen andHras^(val12), which minimally transform the cells but stimulate growthand bypass cellular senescence. Other suitable oncogenes include, e.g.,HPV E7, HPV E6, c-myc, and CDK4 (see also U.S. Pat. No. 5,723,333). Inaddition, the cells can be transduced with an oncogene encodingmammalian telomerase, such as hTRT, to facilitate immortalization.Suitable oncogenes can be identified by those of skill in the art, andpartial lists of oncogenes are provided in Bishop et al., RNA TumorViruses, vol. 1, pp. 1004-1005 (Weiss et al., eds, 1984), and Watson etal., Molecular Biology of the Gene (4^(th) ed. 1987). In some cases theoncogenes provide growth factor-independent and ECM-independent entryinto the cell cycle. Often the oncogenes are dominant oncogenes. Thecells can be analyzed for recombinant oncogene expression by analysis ofoncogene RNA or protein expression. Integration of an oncogene into thegenome can be confirmed, e.g., by Southern blot analysis. Often, theoncogenes are delivered to the cells using a viral vector, preferably aretroviral vector, although any suitable expression vector can be usedto transduce the cells (see, e.g., U.S. Pat. No. 5,723,333, whichdescribes construction of vectors encoding one or more oncogenes andtransduction of pancreas endocrine cells, see also Halvorsen et al.,Molecular and Cellular Biology 19:1864-1870 (1999)).

The vector used to create the cell lines incorporates recombinase sites,such as lox sites, so that the oncogenes can be deleted by expression ofa recombinase, such as the cre recombinase, in the cells followingexpansion (Halvorsen et al., Molecular and Cellular Biology 19:1864-1870(1999)). Deletion of the oncogenes is useful for cells that are to betransplanted in to a mammalian subject. Other recombinase systemsinclude Saccharomyces cerevisiae FLP/FRT, lambda att/Int, R recombinaseof Zygosaccharomyces rouxii. In addition, transposable elements andtransposases could be used. Deletion of the oncogene can be confirmed,e.g., by analysis of oncogene RNA or protein expression, or by Southernblot analysis.

The cultured cells of the invention also express either endogenous orrecombinant PDX-1 having PDX-1 activity, e.g., alleles, polymorphicvariants, and orthologs (see, e.g., Sander et al., J. Mol. Med.71:327-340 (1997)). Endogenous expression of PDX-1 can be induced usingtranscription factors such as hepatocyte nuclear factor 3 beta, which isinvolved in pancreatic β-cell expression of the PDX-1 gene (see, e.g.,Wu et al., Molecular and Cellular Biology 17:6002-6013 (1997)).Recombinant PDX-1 is delivered to the cells using expression vectors,e.g., viral vectors such as retroviral vectors, as described above.

The vectors used to transduce the cells can by any suitable vector,including viral vectors such as retroviral vectors. Preferably, thevector is one that provides stable transformation of the cells, asopposed to transient transformation.

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

GLP-1 receptor agonists, which are administered to the cells of theinvention, include naturally occurring peptides such as GLP-1,exendin-3, and exendin-4 (see, e.g., U.S. Pat. No. 5,424,286; U.S. Pat.No. 5,705,483, U.S. Pat. No. 5,977,071; U.S. Pat. No. 5,670,360; U.S.Pat. No. 5,614,492), GLP-1 analogs (see, e.g., U.S. Pat. No. 5,545,618and U.S. Pat. No. 5,981,488), and small molecule analogs. GLP-1 receptoragonists may be tested for activity as described in U.S. Pat. No.5,981,488. Cells are contacted with a GLP-1 receptor agonist in a timeand amount effective to induce insulin mRNA expression, as describedherein. Typically, the cells are contacted with the GLP-1 receptoragonists for a discrete time period, as the GLP-1 receptor agonist isbelieved to act as a switch for insulin gene expression. Continuousadministration of the GLP-1 receptor agonist is therefore not required.

Immune rejection of grafted cells has previously been a major obstacleto successful islet transplantation. Any universal human donor cell willbe recognized by the immune system as an allograft. However, recentadvances in therapy for allograft rejection may make this less of aconcern (see, e.g., Kenyon et al, Proc. Natl Acad. Sci. USA 96:8132-7(1999)). An advantage of using an immortalized cell line as a source oftransplantable cells is that they can be engineered to exhibit desirablequalities, including avoidance or suppression of host immune responses.

Cell Culture

This invention relies upon routine techniques in the field of cellculture, and suitable methods can be determined by those of skill in theart using known methodology (see, e.g., Freshney et al., Culture ofAnimal Cells (3^(rd) ed. 1994)). In general, the cell cultureenvironment includes consideration of such factors as the substrate forcell growth, cell density and cell contract, the gas phase, the medium,and temperature.

The cells of the invention are grown under conditions that provide forcell to cell contact. In a preferred embodiment, the cells are grown insuspension as three dimensional aggregates. Suspension cultures can beachieved by using, e.g., a flask with a magnetic stirrer or a largesurface area paddle, or on a plate that has been coated to prevent thecells from adhering to the bottom of the dish. In a preferredembodiment, the cells are grown in Costar dishes that have been coatedwith a hydrogel to prevent them from adhering to the bottom of the dish.

For the cells of the invention that are cultured under adherentconditions, plastic dishes, flasks, roller bottles, or microcarriers insuspension are used. Other artificial substrates can be used such asglass and metals. The substrate is often treated by etching, or bycoating with substances such as collagen, chondronectin, fibronectin,and laminin. The type of culture vessel depends on the cultureconditions, e.g., multi-well plates, petri dishes, tissue culture tubes,flasks, roller bottles, and the like.

Cells are grown at optimal densities that are determined empiricallybased on the cell type. For example, a typical cell density for βlox5cultures varies from 1×10³ to 1×10⁷ cells per ml. Cells are passagedwhen the cell density is above optimal.

Cultured cells are normally grown in an incubator that provides asuitable temperature, e.g., the body temperature of the animal fromwhich is the cells were obtained, accounting for regional variations intemperature. Generally, 37° C. is the preferred temperature for cellculture. Most incubators are humidified to approximately atmosphericconditions.

Important constituents of the gas phase are oxygen and carbon dioxide.Typically, atmospheric oxygen tensions are used for cell cultures.Culture vessels are usually vented into the incubator atmosphere toallow gas exchange by using gas permeable caps or by preventing sealingof the culture vessels. Carbon dioxide plays a role in pH stabilization,along with buffer in the cell media and is typically present at aconcentration of 1-10% in the incubator. The preferred CO₂ concentrationtypically is 5%.

Defined cell media are available as packaged, premixed powders orpresterilized solutions. Examples of commonly used media include DME,RPMI 1640, DMEM, Iscove's complete media, or McCoy's Medium (see, e.g.,GibcoBRL/Life Technologies Catalogue and Reference Guide; SigmaCatalogue). Typically, low glucose DME or RPMI 1640 are used in themethods of the invention. Defined cell culture media are oftensupplemented with 5-20% serum, typically heat inactivated, e.g., humanhorse, calf, and fetal bovine serum. Typically, 10% fetal bovine serumis used in the methods of the invention. The culture medium is usuallybuffered to maintain the cells at a pH preferably from 7.2-7.4. Othersupplements to the media include, e.g., antibiotics, amino acids,sugars, and growth factors such as hepatocyte growth factor/scatterfactor.

Pharmaceutical Compositions and Administration

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered (e.g., cell), as well as bythe particular method used to administer the composition. Accordingly,there are a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed., 1989).

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, intraperitoneal,and subcutaneous routes, include aqueous and non-aqueous, isotonicsterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. In the practice of thisinvention, compositions can be administered, for example, by directsurgical transplantation under the kidney, intraportal administration,intravenous infusion, or intraperitoneal infusion.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets. The dose administered to a patient, inthe context of the present invention should be sufficient to effect abeneficial therapeutic response in the patient over time. The dose willbe determined by the efficacy of the particular cells employed and thecondition of the patient, as well as the body weight or surface area ofthe patient to be treated. The size of the dose also will be determinedby the existence, nature, and extent of any adverse side-effects thataccompany the administration of a particular vector, or transduced celltype in a particular patient.

In determining the effective amount of the cells to be administered inthe treatment or prophylaxis of conditions owing to diminished oraberrant insulin expression, the physician evaluates cell toxicities,transplantation reactions, progression of the disease, and theproduction of anti-cell antibodies. For administration, cells of thepresent invention can be administered in amount effective to providenormalized glucose responsive-insulin production and normalized glucoselevels to the subject, taking into account the side-effects of the celltype at various concentrations, as applied to the mass and overallhealth of the patient. Administration can be accomplished via single ordivided doses.

Assays for Modulators of B-Cell Function

A. Assays

Assays using the cultured cells of the invention can be used to test forinhibitors and activators of β-cell function, e.g., insulin productionand/or glucose responsive insulin production. Such modulators are usefulfor treating various disorders involving glucose metabolism, such asdiabetes and hypoglycemia. Treatment of dysfunctions include, e.g.,diabetes mellitus (all types); hyperinsulinism caused by insulinoma,drug-related, e.g., sulfonylureas or excessive insulin, immune diseasewith insulin or insulin receptor antibodies, etc. (see, e.g., Harrison'sInternal Medicine (14^(th) ed. 1998)).

Modulation is tested using the cultures of the invention by measuringinsulin gene expression, optionally with administration of glucose,e.g., analysis of insulin mRNA expression using northern blot, dot blot,PCR, oligonucleotide arrays, and the like; and analysis of insulinprotein expression (preproinsulin, proinsulin, insulin, or c-peptide)using, e.g., western blots, radio immune assays, ELISAs, and the like.Downstream effects of insulin modulation can also be examined. Physicalor chemical changes can be measured to determine the functional effectof the compound on β cell function. Samples or assays that are treatedwith a potential inhibitor or activator are compared to control sampleswithout the test compound, to examine the extent of modulation.

B. Modulators

The compounds tested as modulators of β-cell function can be any smallchemical compound, or a macromolecule, such as a protein, sugar, nucleicacid or lipid. Typically, test compounds will be small chemicalmolecules and peptides. Essentially any chemical compound can be used asa potential modulator or ligand in the assays of the invention, althoughmost often compounds can be dissolved in aqueous or organic (especiallyDMSO-based) solutions are used. The assays are designed to screen largechemical libraries by automating the assay steps and providing compoundsfrom any convenient source to assays, which are typically run inparallel (e.g., in microtiter formats on microtiter plates in roboticassays). It will be appreciated that there are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al, Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville,Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

The assays can be solid phase or solution phase assays. In the highthroughput assays of the invention, it is possible to screen up toseveral thousand different modulators or ligands in a single day. Inparticular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 96 modulators. If 1536 well plates are used, thena single plate can easily assay from about 100- about 1500 differentcompounds. It is possible to assay many plates per day; assay screensfor up to about 6,000, 20,000, 50,000, or 100,000 or more differentcompounds is possible using the integrated systems of the invention.

Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

“Inducing insulin gene expression” refers to increasing, in a cell orculture of cells, the level of expression from the insulin gene by atleast about 25%, preferably 50%, 100%, 500%, 1000%, 5000%, or higher, ascompared to a negative control culture. Insulin gene expression can bemeasured by methods known to those of skill in the art, e.g., bymeasuring insulin RNA expression, preproinsulin, proinsulin, insulin, orc-peptide production, e.g., using PCR, hybridization, and immunoassays.

Cells that “secrete insulin in response to glucose” are cells or a cellculture that, in comparison to a negative control (either non-insulinresponsive cells or insulin responsive cells that are not exposed toglucose), have increased insulin secretion in response to glucose of atleast about 25%, preferably 50%, 100%, 500%, 1000%, 5000%, or higherthan the control cells (measured as described above).

“Culturing cells” so that the cells are “in contact with other cells inthe culture” refers to culture conditions that allow cell to cellcontact. Under such conditions, many but not all cells are in contactwith one or more other cells of the culture. Such conditions includeculturing the cells on a solid surface, such as a plate or a bead, orculturing the cells in suspension.

“Endocrine pancreas cells” refers to cells originally derived from anadult or fetal pancreas, preferably islet cells. “Cultured” endocrinepancreas cells refers to primary cultures as well as cells that havebeen transformed with genes such as an oncogene, e.g., SV40 T antigen,ras, or a telomerase gene (e.g., hTRT).

A “GLP-1 receptor agonist” refers to GLP-1, a GLP-1 analog, or anaturally occurring peptide that binds to the GLP-1 receptor (e.g.,exendin-3 or -4), thereby activating signal transduction from thereceptor.

“Culturing” refers to growing cells ex vivo or in vitro. Cultured cellscan be non-naturally occurring cells, e.g., cells that have beentransduced with an exogenous gene such as an oncogene or a transcriptionfactor such as PDX-1. Cultured cells can also be naturally occurringisolates or primary cultures.

A “stable” cell line or culture is one that can grow in vitro for anextended period of time, such as for at least about 50 cell divisions,or for about 6 months, more preferably for at least about 150 celldivisions, or at least about ten months, and more preferably at leastabout a year.

“Modulating β-cell function” refers to a compound that increases(activates) or decreases (inhibits) glucose responsive insulin secretionof an endocrine pancreas cell. Glucose responsive insulin secretion canbe measured by a number of methods, including analysis of insulin mRNAexpression, preproinsulin production, proinsulin production, insulinproduction, and c-peptide production, using standard methods known to ofskill in the art. To examine the extent of modulation, cultured cellsare treated with a potential activator or inhibitor and are compared tocontrol samples without the activator or inhibitor. Control samples(untreated with inhibitors or activators) are assigned a relativeinsulin value of 100%. Inhibition is achieved when the insulin valuerelative to the control is about 90%, preferably 75%, 50%, and morepreferably 25-0%. Activation is achieved when the insulin value relativeto the control is 110%, more preferably 125%, 150%, and most preferablyat least 200-500% higher or 1000% or higher.

“Transduction” refers to any method of delivering an exogenous nucleicacid, e.g., an expression vector, to a cell, including transfection,lipofection, electroporation, viral transduction, microinjection,particle bombardment, receptor mediated endocytosis, and the like.

A diabetic subject is a mammalian subject, often a human subject, thathas any type of diabetes, including primary and secondary diabetes, type1 NIDDM-transient, type 1 IDDM, type 2 IDDM-transient, type 2 NIDDM, andtype 2 MODY, as described in Harrison's Internal Medicine, 14th ed.1998.

“Expressing” a gene refers to expression of a recombinant or endogenousgene, e.g., resulting in mRNA or protein production from the gene. Arecombinant gene can be integrated into the genome or in anextrachromosomal element.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al, Nature 348:552-554(1990))

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc. (1985)). Techniques for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen, e.g., ELISA, western blot, RIA, immunoprecipitation andthe like. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins    (1984)).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

In one embodiment of the invention the expression vector is a viralvector, preferably one that integrates into the host cell genome, suchas a retroviral vector, or an adeno-associated viral vector. Examples ofretroviruses, from which viral vectors of the invention can be derived,include avian retroviruses such as avian erythroblastosis virus (AMV),avian leukosis virus (ALV), avian myeloblastosis virus (ABV), aviansarcoma virus (ACV), spleen necrosis virus (SNV), and Rous sarcoma virus(RSV); non-avian retroviruses such as bovine leukemia virus (BLV);feline retroviruses such as feline leukemia virus (FeLV) or felinesarcoma virus (FeSV); murine retroviruses such as murine leukemia virus(MuLV), mouse mammary tumor virus (MMTV), murine sarcoma virus (MSV),and Moloney murine sarcoma virus (MoMSV); rat sarcoma virus (RaSV); andprimate retroviruses such as human T-cell lymphotropic viruses 1 and 2(HTLV-1, 2) and simian sarcoma virus (SSV). Many other suitableretroviruses are know to those of skill in the art. Often the virusesare replication deficient, i.e., capable of integration into the hostgenome but not capable of replication to provide infective virus.

In another embodiment of the invention, the vector is a transient vectorsuch as an adenoviral vector, e.g., for transducing the cells with arecombinase to delete the integrated oncogenes.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example I Preparation of Immortalized Human Pancreatic β-Cell Lines

To develop cell lines from human β-cells, islet preparations wereenriched for β-cells based on flavin adenine dinucleotide (FAD)autofluorescence prior to retroviral infection, as described below (FIG.1). Immunohistochemical analysis showed that cells with low FADautofluorescence contained mostly a with a small number of β cells,while cells with high FAD autofluorescence (selected cells in box)contained 96% insulin-positive cells. Glucagon but not insulin wasdetectable by RIA in culture medium from the low FAD population, whileinsulin but not glucagon was detectable in culture medium from high FADcells.

The 96% pure insulin-positive population was used to create the βlox5cell line by infection with the LTPRRNLlox retroviral vector expressingSV40 T antigen and H-ras^(val12) (Halvorsen et al., Molecular andCellular Biology 19:1864-1870 (1999)). This cell line exhibits anextended lifespan and is immortalized by infection with a retroviralvector expressing the hTRT telomerase gene (Halvorsen et al., Molecularand Cellular Biology 19:1864-1870 (1999)). Insulin was detectable at lowlevels shortly after selection in G418, but was rapidly lost (FIG. 2A,lane 9). Furthermore, βlox5 does not express other pancreatic hormones(FIG. 2C).

To isolate the cells, a single cell suspension was prepared from anadult islet preparation of 80% purity. Islets with a diameter of 50-100μm were handpicked under direct vision using a stereoscope afterstaining with dethrone (60 μmol/L). Picked islets were incubatedovernight in RPMI containing 2.5 mM glucose, then dissociated intosingle cells using non-enzymatic dissociating solution (Sigma Corp., StLouis, Mo.) and shaking in a 37° C. water bath at 60 cycles/minute aspreviously described. The resulting single cell suspension was analyzedin a fluorescence-activated cell sorter (Facstar Plus, Becton Dickinson,Mansfield, Mass.) and particle flavin adenine dinucleotide (FAD)autofluorescence (510-550 nm) was plotted against forward light scatter.Two islet cell populations became apparent when FAD autofluorescence(510-550 nm) was plotted against forward light scatter, as previouslydescribed for rat islets.

Highly autofluorescing cells were sorted into sterile Hanks BSS, washedand resuspended in RPMI containing 5 mM glucose and 10% FBS at aconcentration of 2×10⁶ cells/ml. Cells were aggregated into cellclusters by placing 50 μl droplets in a petri dish, gently inverting,and incubating overnight. Thereafter monolayers were initiated from thecell aggregates as previously described for adult islets on HTB-9 matrixin the presence of HGF/SF. After 2-3 days, when the cells were observedto be dividing, they were infected with high titer retroviral vector.

Example II Growth Factor Expression in βlox5 Cells

The expression of some of the transcription factors that are importantin establishing and maintaining β-cell differentiated function wasexamined (FIG. 3). Most significantly, PDX-1, a homeodomaintranscription factor that is required for β-cell development andfunction, is not expressed in βlox5, as shown by RT-PCR (not shown) orEMSA (FIG. 3A, lane 2). This result differs from growth-stimulatedprimary cells, where PDX-1 continues to be expressed (Beattie et al.,Diabetes 48:1013-9 (1999)). BETA2, a bHLH factor important inneurogenesis as well as β-cells is not expressed in βlox5. CREB, whichplays a critical role in cAMP responsiveness in β-cells, is also notexpressed (FIG. 3D). However, Pax6, which is expressed in all endocrinecells, was expressed at high levels in βlox5, consistent with an originfrom primary endocrine cells. RIPE3b, a factor that binds to an elementin the insulin promoter but that has not yet been isolated, is presentas detected by EMSA (not shown). Overall, some, but not all, of thetranscription factors that play important roles in establishing andmaintaining β-cell function are expressed in βlox5 cells.

Example III Expression of Recombinant PDX-1 in βlox5 Cells

Previously, it has been shown that introduction of PDX-1 into a cellline, TRM-6, derived from human fetal islets, resulted in an increase insomatostatin gene expression. PDX-1 also acted synergistically withcell-cell contact to further increase the level of somatostatinexpression about 1,000-fold above the cells not expressing PDX-1, to alevel similar to that found in normal human islets. To determine whetherβlox5 could be induced to express pancreatic hormones, recombinant PDX-1was expressed in βlox5 using a retroviral vector. While this resulted infunctional PDX-1 expression in the cells (FIG. 3A, lane 3), in contrastto TRM-6, βlox5 did not respond to PDX-1 and cell-cell contact with anincrease in hormone expression (FIG. 2A, lane 3, FIG. 2C, lane 3),despite the fact that PDX-1 expression in βlox5 resulted in functionalprotein as evidenced by its ability to bind DNA in EMSA (FIG. 3A, lane3). Therefore, other factors were examined for their ability to inducehormone expression in βlox5 cells expressing PDX-1. Activin andbetacellulin have been reported to be able to induce endocrinedifferentiation in cell lines and primary cells (Mashima et al.,Diabetes 48:304-9 (1999), Huotari et al., Endocrinology 139, 1494-9(1998), Mashima et al., J Clin Invest 97:1647-54 (1996), Yamaoka et al,J Clin Invest 102:294-301 (1998)). However, they had no effect onhormone expression in βlox5 cells (data not shown).

Example III Treatment of PDX-1 Expressing Cells with GLP-1

GLP-1, a small peptide cleaved from the proglucagon molecule, is aninsulin secretogogue Drucker et al., Diabetes 47:159-69 (1998)). Ahomolog of GLP-1 derived from Gila lizards, exendin-4, has recently beenfound to stimulate rodent β-cell differentiation in vitro and in vivo(Xu et al., Diabetes 48:2270-2276 (1999), Zhou et al., Diabetes48:2358-66 (1999). Therefore, exendin-4 was tested for its ability tostimulate β-cell differentiation in βlox5. 10 nmolar exendin-4 wasadministered to the cells for four days, and then removed from theculture medium.

By itself, exendin-4 had no effect on hormone expression in βlox5 (FIG.2A, lane 8). However, when βlox5 cells expressing PDX-1 were grown insuspension culture as three-dimensional aggregates in the presence ofthe exendin-4, a substantial amount of insulin mRNA was detectable (FIG.2A, lane 2). The nature of the interaction between exendin-4, cell-cellcontact, and PDX-1 is evident from the fact that no hormone mRNA wasdetectable with any one or two of the three inducing factors (FIG. 2A).Quantitative RT-PCR revealed that the level of insulin mRNA in inducedβlox5 cells was as that found in adult islets (FIG. 2B). Islet amyloidpolypeptide (IAPP) mRNA, a hormone that is co-secreted with insulin, wasalso expressed in induced βlox5 cells (FIG. 2C, lane 2). However, theother major pancreatic hormones, glucagon, somatostatin, and pancreaticpolypeptide (PP), were not induced (FIG. 2C).

Example IV Expression of Pancreatic Hormones in PDX-1 Expressing βlox5Cells

The expression of transcription factors important in β-cell developmentwas examined to determine whether they had been induced by PDX-1,cell-cell contact, and exendin-4. BETA2 was induced only by thecombination of all three inducing factors (FIG. 3C). Interestingly,CREB, which plays an important role in the cAMP response of β-cells, wasinduced by exendin-4 alone, independently of cell-cell contact and PDX-1(FIG. 3D). GLP-1 has been reported to increase cAMP levels and this hasbeen thought to be a major mechanism by which GLP-1 acts as an insulinsecretogogue (Serre et al., Endocrinology 139:4448-54 (1998)). This isthe first report that activation of the GLP-1 receptor can induce CREBexpression, suggesting that GLP-1 may act at multiple levels inpromoting cAMP signaling. RIPE3b, a factor that plays a critical role ininsulin gene expression but that has not yet been cloned, is expressedin βlox5 irrespective of PDX-1, cell-cell contact, or exendin-4 (FIG.3E).

Example V Glucose Responsiveness

A hallmark of functional β-cells is the ability to secrete insulin inresponse to blood glucose. Primary β-cells store large quantities ofinsulin and secrete it in response to a variety of physiologicalstimuli, most significantly extracellular glucose. Induced βlox5 cellshave an insulin content of 6 picograms per microgram DNA at 1.6 mMglucose. Intracellular insulin is easily detectable byimmunohistochemistry (FIG. 4A). Media from induced βlox5 cells containsmature insulin that comigrates with bovine insulin (FIG. 4B). A majorcomponent of the glucose-sensing apparatus is glucokinase (Matschinskyet al., Diabetes 47:307-315 (1998)). Induced βlox5 cells expressglucokinase mRNA, while uninduced cells do not (FIG. 5A). The extent towhich βlox5 cells exhibit glucose-responsive insulin secretion wastested by culturing βlox5 cells in different concentrations of glucoseand measuring insulin release in the culture medium by RIA (FIG. 5B).Half-maximal secretion occurred at approximately 11 mM, similar tonormal adult islets.

Example VI Transplantation of Glucose Responsive Cells in vivo

βlox5 cells expressing PDX-1 were transfected with an adenoviral vectorexpressing cre, to excise the recombinant SV40 T antigen andH-ras^(val12) genes, as described in Halvorsen et al., Molecular andCellular Biology 19:1864-1870 (1999). The cells had been previouslystimulated with exendin-4, as described above. The cells weretransplanted under the kidney capsule of nude mice for three months. Oneto ten million cells were transplanted in a mice that weighed about 25grams. After three months, the mice were bled for c-peptide and thegrafts were harvested. C-peptide was measured according to standardprocedures using RIA (FIG. 6). These cells exhibit glucose-responsiveinsulin secretion.

1. A method for inducing insulin gene expression in cultured endocrinepancreas cells, the method comprising the steps of: (i) expressing aPDX-1 gene in endocrine pancreas cells that have been cultured underconditions such that the cells are in contact with other cells in theculture; and (ii) contacting the cells with a glucagon-like peptide-1(GLP-1) receptor agonist, thereby inducing insulin gene expression inthe cells.
 2. A method of treating a diabetic subject by providing tothe subject cells that secrete insulin in response to glucose, themethod comprising the step of administering to the subject an effectiveamount of cells cultured according to the method of claim
 1. 3. A methodof treating a diabetic subject by providing to the subject cells thatsecrete insulin in response to glucose, the method comprising the stepsof: (i) contacting a culture of endocrine pancreas cells expressing arecombinant polynucleotide encoding PDX-1 with a GLP-1 receptor agonist,wherein the cells have been cultured under conditions such that thecells are in contact with other cells in the culture; and (ii)administering the cells to the subject, thereby providing to the subjectcells that secrete insulin in response to glucose.
 4. The method ofclaim 3, wherein the diabetic subject is a human.
 5. The method of claim4, wherein the subject has Type I insulin dependent diabetes.
 6. Themethod of claim 3, wherein the GLP-1 receptor agonist is a GLP-1 analog.7. The method of claim 3, wherein the GLP-1 receptor agonist has anamino acid sequence of a naturally occurring peptide.
 8. The method ofclaim 7, wherein the GLP-1 receptor agonist is GLP-1, exendin-3, orexendin-4.
 9. The method of claim 3, wherein the cells are cultured asaggregates in suspension.
 10. The method of claim 1, wherein the methodfurther comprises: (a) transfecting the cells with a vector comprisingat least two oncogenes and at least one inducible promoter, wherein theexpression of each oncogene is under the transcriptional control of aninducible promoter and wherein said oncogenes are capable of expressionin the cells to induce their multiplication; (b) allowing thetransfected cell after step (a) to divide at least once after step (a);and (c) removing said oncogenes.
 11. The method of claim 10, wherein thevector further comprises a pair of genetic elements flanking theoncogenes, wherein the genetic elements comprise recombination sites.12. The method of claim 11, wherein the genetic elements comprise loxsites.
 13. The method of claim 12, wherein the oncogenes are removedfrom the cells by introducing the Cre recombinase into the cells. 14.The method of claim 1, wherein the cultured endocrine pancreas cells arehuman cells.